Accepted Manuscript

A systematic reconsideration on proteases

Poorani Gurumallesh, Kamalini Alagu, Baskar Ramakrishnan,

Shanmugaprakash Muthusamy

PII: S0141-8130(18)35199-7
DOI: https://doi.org/10.1016/j.ijbiomac.2019.01.081
Reference: BIOMAC 11514
To appear in: International Journal of Biological Macromolecules
Received date: 3 October 2018
Revised date: 3 January 2019
Accepted date: 18 January 2019

Please cite this article as: Poorani Gurumallesh, Kamalini Alagu, Baskar Ramakrishnan,
Shanmugaprakash Muthusamy , A systematic reconsideration on proteases. Biomac
(2018), https://doi.org/10.1016/j.ijbiomac.2019.01.081

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A systematic reconsideration on proteases

Poorani Gurumallesh, Kamalini Alagu, Baskar Ramakrishnan*, Shanmugaprakash Muthusamy

Department of Biotechnology, Kumaraguru College of Technology, Coimbatore, India

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Corresponding author

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Dr. R. Baskar

Associate Professor

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Kumaraguru College of Technology NU
Chinnavedampatti, Saravanampatty

Coimbatore – 641049
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Email: baskar.r.bt@kct.ac.in

Phone: +91 9894709273

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+91 422 2661100

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Conflict of Interest: The authors declare no conflict of interest

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Abstract

Proteases are a group of large complex enzyme molecules that perform highly focused proteolysis
functions. A vast quantity of the protease enzymes is predominantly sourced from microbial
fermentation process, although proteases tend to natively present in plant, animals and humans.
Proteases possess a pervasive importance in medical and pharmaceutical sector, because of its enriched
specificity towards biomolecules. They are also actively encompassed in regulating certain
physiological pathways. A distinct territory of human disorders is treated by substrate specific
proteases. Enormous numbers of catalytic activities in habitual metabolism process of a living organism

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are protease dependent. Pilot scale researches and product development in industrial biotechnology
sectors are wholly based on any one of the protease enzymes. The applications of the protease enzymes

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and its economic benefits of being an eco-friendly material are far-reaching. This review presents a
brief overview on the classification and sources of various types of proteases. We describe the essential

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evidences of role of protease in different sectors. The proteases could be a potential relieves to harmful
synthetic chemicals in distinctive industrial processes and thus gains global perception.
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Keywords: Proteases; Categorization; Industrial application.
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1. Overview

The peptide bonds in between amino acids and proteins are hydrolysed by proteases. Around 60%
enzymes in the global market accounts to proteases [1]. The catalytic activity of the protease are
involved in various processes like digestion, apoptosis and so on. Proteases are so much indispensable
in all living organisms for synthesis of necessary biomolecules and to control the size, shape, turnover
and the composition of fundamental proteins[2]. The International protease network states that every
human hold closely 500 proteases accounting to 2% of total genome. However, the metabolic role of
certain proteases in the human system are yet to be studied [3]. The highly specific and limited cleavage

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in the substrate by the protease is one among the foremost metabolic reactions in all of the living
organisms. Hydrolysis by protease is an originator to various biological process such as DNA

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replication and cell cycle [4].

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Proteases are classified by its diverse attributes. Proteases were acquired majorly from various plant
and animal sources before the exploitation of the microbes. In a while, the plants and animals confronted
hitches in its continued existence due to climatic factors and cumulative devastation of natural habitats
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by humans. The ethical issues were on rise upon sacrificing animals to isolate protease. Thereby the
microbes arrive at attention for researchers to produce large scale protease enzyme at high yield rates.
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Almost two-third of the commercial proteases available in the market all over the world belongs to
proteases from microorganisms [5].

Proteases are one of the most experimental enzymes and plays decisive role at different sectors such as
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commercial, pharmaceutical, analytical, diagnostic and effluent abatement. In this review, the
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comprehensive description of classification of protease, sources of protease enzyme and the potential
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utilizations of the enzyme in a variety of industries will be discussed. The overview of the review article
is illustrated in the graphical abstract (Figure 1).
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2. Categorization of Proteases
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The IUBMB Nomenclature Committee states that classification of proteases could be made under
subgroup 4 of group 3 (hydrolases) [6]. The grouping of proteases is based on various strategies such
as the reaction type where protease acts as a catalyst; the nature and the chemical properties of the
catalytic site and the evolution of the protease structure and their relationship [7]. The organisation of
protease based on their specificity or catalytic mechanism was discussed by Agarwal [8]. Protease (E.C
No.3.4) enzyme that hydrolyse the peptide bonds are further sectioned into Exopeptidases (E.C. No
(3.4.11 -3.4.19) and Endopeptidases (E.C. No (3.4.21-3.4.24, 3.4.99) depending upon the type of the
reaction [9].

2.1 Exopeptidases
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Exopeptidases executes its enzyme activity by hydrolysis merely on the nitrogen or carbon terminal
points of the substrate chain entirely made up of polypeptide [10]. Exopeptidases are further sorted
conferring to the specificity such as the size of the fragment, identity of the liberated fragment, terminus
of the origin and the size restriction on the length of the specific peptide chain [8]. Exopeptidases acts
in two forms, namely Aminopeptidase and Carboxypeptidase.

As the name indicates, the aminopeptidases put on its activity at a free N terminal point of the protein
and as a result of digestion it releases a mono amino acid residue, a dipeptide, or a tripeptide [11]. The

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presence of aminopeptidases is reported in a wide variation of microbial species that includes bacteria
as well as fungi, plants and animals. The aminopeptidases are secreted as intracellular and extracellular

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aminopeptidases [12]. Methionine aminopeptidases (MAP) accomplishes crucial cellular functions,
which shares homology with a vast range organism, and functions by cleaving N-terminal methionine

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from freshly synthesized proteins [13].
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The C terminals of the protein chain is majorly influenced by carboxypeptidases. Depending on the
character of the residual amino acid at the active site, carboxypeptidases are clustered under three
classifications: serine carboxypeptidases, cysteine carboxypeptidases, and metallo carboxypeptidases
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[14]. The first generation carboxypeptidases such as pancreatic carboxypeptidase A1 and A2, have their
potentiality exhibited at the process of digestion of food molecules. The other properties revealed by
the carboxypeptidases are their active participation in healing of internal and external wound and blood
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clotting. The classification of proteases is outlined in Table 1.

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Table 1: Categorization of proteases

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TYPE OF EC.NO MECHANISM EXAMPLES

PROTEASE
Aminopeptidases 3.4. Acts on free N-terminus Cleaves one amino Peptidase_M
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of a protein acid and N-terminal 1

Dipeptidases 3.4.13 Hydrolyses the Cleaves two amino Aspartyl
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dipeptide bond at the N- acid and N-terminal dipeptidase

terminus
Tripeptidases 3.4.11 Hydrolyses the Cleaves three amino Lympho
tripeptide bond at the N- acid and N-terminal peptidase
terminus
Carboxypeptidases 3.4.16- Acts on free C-terminus Cleaves one amino Pancreatic
3.4.18 and releases a single acid from C-terminal carboxypepti
amino acid dase A
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Serine type protease 3.4.16 Serine serves as a Cleaves peptide Subtilisin

nucleophilic amino acid bonds with its partial
at active site negative oxygen
atom
Metallo protease 3.4.17 Catalytic mechanism Cleaves the C- Thermolysin
involves a metal, terminus, the
often zinc unbound COOH
group

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Cysteine type 3.4.18 Catalytic mechanism Deprotonation of a Papain
protease involves a nucleophilic thiol followed by

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cysteine thiol nucleophilic attack

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2.2 Endopeptidases
Endopeptidases are proactively involved in causing cleavage in amino acids that are non-terminal [15].
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The endopeptidases are subjected to classification reliant on the chemical kind of the group that is
primarily in charge for the catalytic activity. There are six classes of protease; (i) Cysteine proteases,
(ii) Serine proteases, (iii) Threonine proteases, (iv) Glutamic acid proteases, (v) Aspartic acid proteases
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and (vi) Metalloproteases.

Serine protease are identified by the occurrence of residual serine in the catalytic binding site [16] .
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Serine proteases are sorted into 20 families depending upon their resemblances in their structures.
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Devising a common ancestor, the families are additionally segmented into six class [11]. Every single
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serine protease encloses three residual molecules at their catalytic binding site: aspartate, histidine and
serine. Serine proteases contribute to one third of all known proteases [17]. The mechanism of serine
peptidase is as simple as fragment transfer like other group of enzymes, although in this case the transfer
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is to water [18]. Considering a deep look into the mechanism of serine protease, almost all of them
retain a basic dyad concept in which Lys or His found to be bound with the catalytic Ser. Other serine
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proteases provoke catalysis through novel triads of residues, such as a pair of His residues combined
with the nucleophilic Ser [17].

Aspartic acid protease are the acidic proteases that use an activated water molecule constrained to
aspartate residues at the catalytic site of action [19]. Those proteases are settled into three families
namely retropepsin, pararetroviruses proteins and pepsin. As per the noted activity of aspartic acid
proteases, the functional group of the enzyme would be targeted. While targeting, there is no nucleopilic
attack happening around and also there is absence of formation of covalent substance that was expected
to occur in between the enzyme and a substrate fragment [18]. The examples of potential aspartic acid
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proteases include pepsin, cathepsin and rennin. Aspartic acid proteases are highly active and efficient
at minimal pH range (3 - 4).

Cysteine proteases provide the validated targets to treat a wide range of human disorders. Prokaryotes
and eukaryotes contain proteases that have cysteine at their active site [20]. The catalytic dyad of
cysteine and histidine are the active components that are crucial for cysteine proteases to exhibit its
potentiality. About 20 families of cysteine proteases have been recognized [14]. Cysteine proteases are
broadly divided into four groups based on side chain specificity: (i) papain-like, (ii) trypsin-like with

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preference for cleavage at the arginine residue, (iii) specific to glutamic acid, and (iv) others (20).
Calpains are a type of cysteine proteases which need calcium ions for enzyme activation. 15 isoforms

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of the calpain were reported and 11 of them have been identified in humans. µ-Calpain and m-calpain
are the two types of calpain that have been isolated which differ in their calcium requirement [11].

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Cysteine proteases are active only in the presence of reducing agents such as HCN or cysteine.
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Metalloproteases belong to the diverse group of the catalytic types of proteases. Every single
metalloprotease requires a divalent metal ion to exhibit its proteolytic activity on the specific protease.
There are about 30 families identified so far [21]. Metalloproteases can be divided into four groups
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depending upon the specificity of their action, (i) neutral, (ii) alkaline, (iii) Myxobacter I, and (iv)
Myxobacter II. In one way, metalloprotease is comparable to aspartic peptidases so that they do not
form the covalent intermediate, hence they need a metal ion to exert this effect.
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Proteolytic enzyme with a threonine residue in its active side are said to be threonine proteases. To
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perform catalysis, the threonine proteases use a nucleophile which is primarily a secondary alcohol of
the N-terminal threonine. Enzyme catalysis by threonine protease involves two stages. Initially, the
substrate is targeted by the nucleophile to form acyl-enzyme intermediate, followed by water dependent
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hydrolysis to release the products and free enzymes [22].

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Glutamic proteases are characterized in such a way that; all active site contains a glutamic acid residue.
The first ever elucidated structure of the enzyme was scytalidoglutamic protease, where the active
contained a catalytic dyad (Glutamic acid and Glutamine). Glutamic proteases are acidic in nature and
works better at pH 2. Eqolisin is a potential example of glutamic protease which was isolated from the
fungi Scytalidium lignicola [23]. The common characters shared by the different endoproteases are
given in Table 2.

Table 2: Common characteristics of endopeptidases

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Type of protease Serine protease Cysteine / Thiol Aspartic/ Metallo protease

Trypsin protease Carboxyl
Chymotrypsin µ-Calpain protease
m- Calpain Pepsin
Cathapsin B H L Cathapsin D
EC No 3.4.21 3.4.22 3.4.23 3.4.24
Active site of Serine, Histidine Aspartate/ Aspartate/ Phenyl-alanine/
amino acid and Dispartate Cysteine Cysteine Leucine
2+ 2+
Metal Ions Ca Ca Zn2+, Ca2+

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Optimum pH 6-11 2-3 2-5 5-8
Temperature 50-70 40-55 40-55 65-85

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(ºC)
Molecular weight 18-35 20-37 30-48 19-37

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(KDa) 69-74
Mechanism Endopeptidase Endopeptidase Endopeptidase Endopeptidase
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have an active have an active have an active have an active
center, serine center, cysteine center, Aspartic center, metal ions
involved in involved in involved in (Zn+) involved in
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catalytic process catalytic process catalytic process catalytic process

Application Blood clotting, Apoptosis of Blood pressure Tumor
immunity and breast cancer cells control; metastasis;
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inflammation Digestive Wound healing;

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response; enzyme; Meat Embryonic

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Digestive tenderizer development

enzymes
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3. Sources of Proteases
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The occurrence of protease is reported in all organisms, like prokaryotes, eukaryotes, plants and
animals. The type of protease secreted is completely dependent on the host and its adaptability to its
immediate environment. A large number of factors must be taken into consideration for fermentative
production of protease from the microbial source. The detailed description of different source of
proteases is given below with examples.

3.1 Plants
The plants as a source of proteases has a lot many considerations that include dependency on suitable
climatic conditions and availability of agricultural land for cultivation. Plants respond to the
environment in such a manner that the environmental signals play a crucial role in normal development
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pathways. The time utilization and labour involvement are the factors to be pondered before electing a
plant as a source to extract protease [24]. In plants, proteases are partaking in the events like
mobilization of storage proteins, apoptosis, etc. Proteases are generally inactive proenzyme fragments
at N-terminal ends of a precursor [25]. Predominantly, plant proteases are cysteine endopeptidases and
rarely other proteases are observed [26]. Bromelain, papain, ficin and keratinases are a few of the
illustrious proteases of plant origin.
Miliin, is a serine protease extracted from latex of Euphorbia milii. The protease exhibited maximal
activity around pH 9 and at 35ºC. The molecular weight determined was about 75 kDa. Miliin is broadly

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used as a molluscicidal agent and as antithrombic drugs [27]. The another type of miliin retrieved from
the Euphorbia species is a thiol dependant serine protease. The 79 kDa enzyme demonstrated activity

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at 60ºC and the active pH range is 7.5-11 [28].

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Carnivorous plants also secrete protease that of huge biotechnological and pharmaceutical importance.
Drosera capensis is one such plant that has a large number of genes that codes for cysteine proteases.
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Upon sequence comparison, cysteine proteases from Drosera species showed homology to various
plant proteases [29]. Wrightin, a known serine protease is obtained from the Wrightia tinctoria latex.
The enzyme having a molecular weight of 57.9 kDa is active at pH 7.5-10 and temperature of 70ºC.
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Wrightin is a potential candidate for food industries [30].

Papain is a plant cysteine protease isolated from Carica papaya latex fruits. The papain has broader
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specificity towards substrates because of presence of numerous isoenzymes within it. Papain is active
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between pH 5 – 9 and found to be stable till 80ºC. The papain plays a key role in varied biological
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process and used comprehensively in meat tenderizing industry, drug designing and so on [31]. Carnein
is a serine protease from the latex of Ipomea carnea. The molecular mass is 80.24 kDa and optimal
conditions for high protease activity are pH 6.5 and 65ºC [32]. An aspartic proteases exhibiting different
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physiological roles is known to be purified from the potato leaves [33]. A thiol dependant protease is
reported in crown leaf of pineapple [34] and a neutral protease from the leaves of Raphanus sativus is
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purified and used in various industrial applications [35].

Each plants express protease under different environmental conditions. In parsley leaves, a 70 kDa
serine protease is observed, where the protease activity is found to increase with advancement in
senescence [36]. Similarly, protease from barley and alfalfa establishes degradation of protein only
during the senescence of the foliar region [37]. Cathepsin B, a cysteine protease from a Himalayan plant
Picrorhiza kurrooa is a 39 kDa protein. The protease is expressed with response to phytohormones that
upregulate the leaf senescence [38]. A group of cysteine proteases C1A are secreted in abundance only
during the leaf senescence. This shows how the plants are dependent on the environment and physiology
to express certain proteins [39].
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Bromelain is an industrially important cysteine protease enzyme obtained from the pineapple parts such
as stem and juices. The active range of enzyme is from pH 5 to 9 and optimum temperature is 70°C.
The optimum temperature is found to be lesser than that of papain. A review by Barun on bromelain
demonstrates the potential applications of the cysteine protease in wide range of industries [40]. The
complete information of plant based protease, source, purification strategies and applications are
described in a review article by Cecilia and Xavier [18].

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3.2 Animals

Very few economically important proteases are originated from animal species. Rennin, chymotrypsin,

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pancreatic trypsin, pepsins are the animal proteases that play a fundamental role in digestive process.
Animal proteases are isolated in bulk quantities depending on the existence of livestock slaughter. The

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extraction is however governed by various policies categorized under political and agricultural sectors
[24]. In order to look for an alternative to minimise the animal slaughter, recombinant DNA technology
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would be performed for massive production of animal proteases. Also, the animal proteases gain
attention to be a bio-control measures for insect pests.
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A serine protease, Trypsin is the main intestinal digestive enzyme present widely in vertebrates. Trypsin
is an important biomolecule that could hydrolyse the proteins present in food. Trypsin acts on the
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carboxyl end of the amino acids lysine or arginine. Trypsin from bovine and porcine sources is of 23.3
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kDa molecular weight and works best at 37ºC. Trypsin is extensively used in a tissue culture lab for the
process of harvesting the cells. Apart from this, trypsin is used as medicinal and food industries [41].
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Chymotrypsin is yet another serine protease found in pancreatic juice of an animal. The mechanism of
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action is that, chymotrypsin attacks the unreactive carbonyl with a help of a nucleophile. Chymotrypsin
does not exhibit allosteric effects. For diagnostic and analytical purposes pure chymotrypsin which is
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an expensive enzyme is utilized. Chymotrypsin exerts its specificity for hydrolysis of amino acids such
as phenylalanine, tyrosine, or tryptophan [42]. Chymotrypsin is present in precursor form
chymotrypsinogen. Through a cascade of processes, the inactive precursor is converted into
chymotrypsin by trypsin. A large volume de-allergenizing of milk protein is conceded by chymotrypsin.

An acidic aspartyl protease called Pepsin is existing in the stomachs of almost all vertebrates. The
precursor of the pepsin is a zymogen namely pepsinogen. Hydrochloric acid from the parietal cells is
required to activate the zymogen. Pepsin potentially cleaves the amino acids such as phenylalanine,
tryptophan and tyrosine at the N-terminal side. Pepsin has been extracted from Japanese monkey, goat,
rat and rabbit. A review of protease from fish is given in detail by Lisha [43].
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3.3 Microbes

Microbial proteases contribute to nearly two-third of industrially essential proteases. Only certain
microorganisms manufacture proteases with anticipated qualities. The growth and maintenance of the
host microbes when compared to plant and animals is quite stress-free and less time consuming. Some
microorganism has their natural capability to secrete protease by themselves without any induction.
Proteases are produced in large volume by fermentation process of microorganisms in a planned

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bioreactor. Two approaches, solid state and submerged fermentation are exercised to extract protease
from microbes. Among the two processes, solid state fermentation is preferred more, because of low

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consumption of energy. The protease and its characters extracted from different microorganisms are
outlined in Table 3.

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Table 3: Microbial proteases from bacteria and its characteristics
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Micro - Source of Protease Purification Optimal Application Ref.
organism Isolation Type strategy conditions
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Bacillus Gut of Chymo Crude protein pH 7.8 Digestion [44]

subtilis Helicoverp trypsin like Temp of
RTSBA 6 a armigera protease 35ºC proteinacio
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us food
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Bacillus Agro – De-hairing ASP, Sephadex PH 6-9 Detergent [45]

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cereus strain industrial alkaline G-75 column Temp and leather

AT residues protease chromatography 50ºC industry
and cow
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dung
Bacillus Saltern Alkaline Crude Protein pH 8 - [46]
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licheniformis sediments proteases Temp

37ºC
Bacillus Soil Thermostabl ASP, DEAE – pH 7.5 De-hairing [47]
megatarium e alkaline cellulose ion Temp of skin
proteases exchange 50ºC
chromatography
and Sephadex
G-200 gel
filtration
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Bacillus Mangroves Alkaline ASP, SDS-Page pH 7 Detergent [48]

circulans proteases Temp additive
BM15 40ºC
Bacillus Pigeon pea Cellulase Crude protein pH 11 Food [49]
species JB-99 waste as free Temp industry
substrate thermostable 70ºC
protease
Pseudomona Tannery Salt tolerant ASP pH 9 Leather [50]

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s aeruginosa saline protease Temp industry
strain BC1 waste water 30ºC

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Listeria Degraded Alkaline Crude Protein pH 9 Therapeutic [51]
monocytogen meat of Protease Temp application

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es cow 30ºC s
Bacillus Soil of a Organic Crude Protein pH 10 Potential [52]
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cereus strain wood solvent Temp use in
146 factory tolerant 37ºC industries
alkaline
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protease
Halotolerant Coastal Metalloprote ASP and pH 9 Stain [53]
alkaliphilic sediment of ase Dialysis Temp removal
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Bacillus Goa 37ºC capability

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cereus CS1
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Geobacillus Sewage Thermoactiv Crude Protein pH 8-9 - [54]

caldoproteol sludge e Temp
yticus extracellular 70ºC
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protease
Bacillus - Serine Sephadex G-200 pH 9 Blood stain [55]
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laterosporus protease gel filtration; Temp removal

DEAE cellulose 75ºC
chromatography
Pseudomona Meat waste Alkaline Crude Protein pH 9 - [56]
s fluorescens contaminat protease Temp
e soil 37ºC

The microbes that are employed in fermentative production of protease are briefly explained in a review
article by Kanupriya et al [57]. Protease producing microorganisms are isolated from various sources
like soil, vegetable wastes, milk processing plant [58], and poultry waste contaminated soil [59] and so
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on. Every microorganism needs an optimized growth media and environmental conditions to secrete
the protease enzyme. Once secreted, a series of downstream processing steps must be carried out in
order to obtain pure fraction of the protease enzyme. A brief outline of the protease purification steps
is demonstrated in figure 2.

The next group of microbes to be focused for protease production are the fungi. Filamentous fungi have
an extraordinary capability to secrete functionally active proteins. Trichoderma and Aspergillus are two
dominant fungal strains utilized for industrial protease production. Pleurotus citrinopileatus is an edible

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mushroom that acts as a rich source of alkaline protease [60]. The other fungal species involved in
protease production are described in Table 4.

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Table 4: Microbial proteases from fungi and its characteristics

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Microorganism Source of Protease Purification Optimal Applications Ref.
Isolation Type strategy conditions
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Rhizopus oryzae - Aspartic Anion pH 3.4 Food [61]
protease exchange Temp processing
chromatogra 75ºC industry
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phy
Lentinus citrinus Ligno Neutral Crude pH 7 Food [62]
cellulosic protease protein Temp industry
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waste 50ºC
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Rhizomucor Local Rennet Crude Temp Milk [63]

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nainitalensis isolates protein 40ºC coagulation

Engyodontium Marine Alkaline ASP ion- pH 11 Detergent [64]
album sediment protease exchange Temp industry
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chromatogra 60ºC
phy
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Beauveria felina Soy Alkaline Crude pH 9 Hydrolysis of [65]

protein protease protein Temp soy protein
isolate 55ºC
Penicillium Soil Alkaline Crude pH 8 Detergent [66]
godlewskii SBSS protease protein Temp industry
25 35ºC

Apart from the above mentioned fungi species there are lot many fungal sources that secrete
commercially important protease enzyme. That includes Mucor species [67], Aspergillus clavatus [68],
Penicillium chrysogenum [69] and so on. Proteases from certain actinomycetes also comes under
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microbial proteases. Actinomycetes are a group of bacteria with morphology similar to the fungi.
Streptomyces nogalator is a protease producing actinomycete, isolated from the soil of leather and hair
dumping areas. The enzyme was found to be useful in depilation of goat skin [70]. Another alkali-
thermotolerant protease was isolated from Streptomyces sp DP2, which exhibited maximal activity over
at pH 8 and possessed thermostability at 90ºC [71].

4. Recent advances and strategies to improvise protease production and yield

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Periodically, gene knockdown and genetic manipulation have been undertaken through various
technologies. There are certain ineffectiveness and drawbacks on adopting those conventional

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methodologies as we do not possess any control over prevailing protein production levels. In order to
achieve strategic enzyme production, synthetic biology tools are to be hired. Among the tools, CRISPR

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– Clustered Regularly Interspaced Short Palindromic Repeats, are the most effective with high
potentiality in achieving gene modification [72]. The entire CRISPR system is composed of two major
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components such as Cas9, which is a double stranded DNA nuclease and a personalised cofactor
(synthetic guide RNA) that could selectively target genes to induce DNA breakage. Bacillus subtilis
ATCC 6051 is an active strain involved in the production of industrial enzymes with its outstanding
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ability in secreting proteins. The major hurdles experienced by the researchers on using this strain are
insignificant transformation efficiency. Under such positions, CRISPR/Cas9 system was preferred by
Zhang et.al [73]. Future studies on protease production can think of using CRISPR/Cas9 system to
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enhance gene editing, thereby improvising gene expression of industrially important proteases.
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A lot of computer based software are user friendly to perform designing the synthetic DNA segments.
Huge number of codon optimization algorithms are available, through which the DNA construct can be
optimized for high level of expression of protein in selected host organism. Gene Designer is a software
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available for molecular biologists to expand research in synthetic biology [74]. Another important thrust
area in sequencing related segment is database mining. Metagenomics is a perfect tool through which
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DNA could be isolated from environmental micro-organisms and the sequence information is
processed. The technique is followed by screening of open reading frames from the isolated sample in
order to identify any novel genes that could code for a novel enzyme [75]. Through metagenomics
screening, a lot many enzymes have been identified and isolated with novel bio-catalytic activity. There
was a report stating that, by using metagenomics technology a novel protease enzyme had been
identified by Waschkowitz [76]. Developing techniques like metaproteomics and metatranscriptomics
are the potential fields on which researchers could put more attention in order to find a novel enzyme
or a novel source.
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Compared to older days, more number of enzyme engineering techniques have emerged to improvise
the yield and the performance of the protein fraction. A detailed description about these factors were
discussed by Lutz and Stefan [77]. A few among them are phage display [78] and cell free system for
enzyme production [79]. Manufacturing enzymes is a sustainable process that are highly associated
with synthesis of chemicals. A noteworthy effort is being made every day to explore new enzymes and
also in enhancing the efficiency of existing enzymes [80]. One of the major hiccups in analysing
enzymatic reactions are, the substrate and products are not being distinguished separately by optical

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spectroscopy. To overcome such situations, researchers are following separation based assays so that
the product and the substrate are detected individually. Capillary electrophoresis has been proved to be

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a better separation technique than chromatography on performing differentiation of substrate and
product [81]. Though, there are several upcoming and existing techniques in enzyme production and

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usage, the demand for enzyme still persists in food and pharmaceutical sector. The enzyme synthesized
so far, could be insufficient to meet the need of the large growing population, and the impact of the
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enzyme market on developing countries like India is enormous [82].

The last decade has witnessed an alarming achievements in science and technology with extensive
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potential of nanotechnology. Their convoluted characters like high surface-to-volume ratio, stability,
higher adsorption and catalysis efficiency make them a suitable platform for achieving target product
various disciplines in science. In the second half of 1980s, nanotechnology has been applied in enzyme
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production and characterisation. Chiefly, the nanoparticles serve as a matrix for immobilizing the
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enzyme biomolecules because of their higher surface area and strong adsorption forces [82].
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Immobilisation, on the whole improves the stability of the enzyme and also safeguards protein from
unfolding. Another very important criteria of using immobilised enzymes are, the enzyme molecules
behave as free enzymes and exposes Brownian movement in aqueous environment, averts the
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aggregation of enzymatic biomolecules thereby enriching enzyme activity. Numerous types such as
metal oxide nanoparticles, polymeric nanoparticles, metal nanoparticles and magnetic nanoparticles
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serves as a base support material with extraordinary properties as an enzyme protectant. The enzyme
molecule is attached to the nanoparticles by any of the four mechanisms; a) Conjugation to the surface
of the nanoparticle directly; b) Adsorption by electrostatic forces; c) Affinity oriented protein
conjugation and d) Surface attachment to reformed nanoparticles by covalence. The detailed description
of the processes was mentioned by Razi Ahmad and Meryam Sardar [75].

5. Selected applications of proteases

5.1 Detergent Industry

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The statistics states that nearly 25-30% of the commercial enzymes are the key ingredients in detergent
making. Amylases and proteases are being extensively used in detergent synthesis process to remove
starch and protein based dirt. In 1913, first enzyme based detergent namely Brunus was introduced and
at the present scenario Novozymes and Genencor are the major suppliers of detergent enzymes [83].
The mode of action of cleaning the stains in the cloth by the detergent is depicted in the figure 3.

The Several alkaline proteases such as serine alkaline protease from Micromonospora chaiyaphumensis
are extremely heat stable and increased thermoactivity was observed in presence of calcium ions [84].

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These properties make them ideally fit for applications in detergent making. More recently, intracellular
serine protease from Bacillus megaterium was very efficient in hydrolysing casein and haemoglobin

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[85]. Keratinolytic proteases are recently involved, thereby breaking down the major bottlenecks in
protease based detergent production and marketing [86]. The researchers are focusing on developing

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detergent with a combination of enzymes to improve the performance. A study states that destaining
ability was high when mannanase and protease are coproduced and used together in a detergent powder
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[87]. Generally, protease enzyme powder would be produced and mixed with dry ingredients in
detergent making process. Heat sensitive ingredients are added just prior to spray drying process. The
outline of detergent making is illustrated in figure 4.
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5.2 Leather industry

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Dehairing is an important process in leather industry and usage of chemicals such as sodium sulphide
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leads various pollution related issues. The protease based dehairing process is eco-friendly and exhibit
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favourable outcomes such as strength and quality of the product. Preceding the decision of the protease
for dehairing, a through screening must be done to obtain substrate specific enzyme thereby not
degrading other substrates [88]. The usage of various protease at different steps of tanning operations
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are depicted in figure 5.

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5.3 Food industry

To hydrolyse the caseins, proteases are added to milk in cheese production process. There has been
serious hunt to find an alternative for usage of rennet in cheese making due to various limitations. Plant
based proteases are identified as a potential source with having potential as that of rennet [89]. The
enzymes used in cheese production recently and their sources are shown in table 5 and the methodology
is shown in the figure 6.

Table 5 Recent proteases in cheese making

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Protease type Source Reference

Aspartic protease Penicillium brevicompactum [90]
Serine protease Foeniculum vulgare [91]
Preprogaline (aspartic protease) Pichia pastoris [92]
Protease Bacillus species P45 [93]
Aspartic protease Withania coagulans [94]
Aspartic protease Cynara scolymus [95]
Aspartic protease Aspergillus foetidus [96]

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Protease Bacillus stearothermophilus [97]
Alkaline protease Solanum melongena [1]

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Cysteine protease (Porcerain B) Dregea sinensis [98]

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The most important aspect for meats from beef, pork and lamb is its tenderness. Meats are stored at
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refrigerated condition and while aging, the proteases present in muscle would break myofibrils thereby
tenderizing it. Cysteine protease calpain 1 (calcium dependant) is an enzyme of prime importance for
softening the meat [99]. A new study explains that the gene coding for protease in Rhizomucor miehei
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(Rmpro-A) is expressed in Pichia pastoris and the product exhibited a high range of tenderization in
pork [100]. Usually, papain served as a potential enzyme for softening the meat over a period of time
and the recent finding shows that Rmpro-A, at a very low concentration exhibits tenderization much
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better than papain. Actinidin is a cysteine protease from chinense kiwifruit and showed improved
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tenderness properties in rabbit and pork muscles [101]. A new identification named proline
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iminopeptidase from lactic acid bacteria is a serine protease that can significantly degrade collagen and
tenderize the meat [102]. Buffaloes are exclusively slaughtered by humankind for meat. Plant
proteolytic enzymes from Cucumis trigonus and Zingiber officinale are nowadays more efficient in
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tenderizing buffalo meat than papain [103].

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The baking industries’ prime research focus is on degrading gluten, in order to improve the quality of
wheat that is involved in dough preparation. A wide variety of plant and microbe based proteases are
utilised in gluten degradation. Neutral proteases are perfect fit for the baking and brewing applications.
Neutrase is a metalloprotease from Bacillus amyloliquefaciens and it is very potent in hydrolysing meat
and gelatin. Neutrase plays a vital role in modification of dough processing for preparation of biscuits
and crackers [104]. Renzetti and Arendt stated that degradation by proteases could be very beneficial
in enhancing the breadmaking ability of brown rice flour [105].

The non-hydrolysed protein content in other milk formulas are allergic and also quite hard to digest by
children. Proteases are very useful in preparing hydrolysed formula milk for infants [106]. More focus
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is being thrown at the production of gluten free beer at the brewing industry. A study shows that
recombinant proline specific prolyl endopeptidase from Sphaerobacter thermophiles is very efficient
in producing gluten free beer. The isolated recombinant protein was added to barley during malting
process resulting in high quality gluten free beer [107].

5.4 Therapeutic applications of Protease

Non-invasive therapeutic procedures are largely evolving in treatment of human diseases and disorders.

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A broad spectrum of medical conditions is being treated through enzymatic degradation of substrate.
The success rate of such treatment is enormous because of specificity of particular enzyme towards

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targeted substrate thereby not harming the surrounding cells. Some of the enzymes are highly selective
and specific even at a very low concentration. Collagenase group of enzymes are matrix

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metalloproteinases that can treat conditions such as Dupuytren’s disease, Peyronie’s disease, wound
healing, burns, glaucoma, intervertebral disc herniation, debridement, degradation of human retained
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placenta, cartilage repair, nipple pain, keloid, vitrectomy, cellulite, chronic total occlusions, gene
delivery and uterine fibroid. These medical conditions and the responsible collagen type used for
treatments are elaborated in detail by Hamzeh Alipour et al [108].
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Enzymes are also formulated as topical applications. Alkaline proteases from Bacillus subtilis acts as
efficient transdermal drug delivery system for dermatological applications [109]. Cysteine proteases are
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best in being a validated target for treating ailments. Cathepsins and calpains are pharmaceutically more
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important cysteine proteases. Cysteine proteases glucoprotein purified from Zingiber montanum proved
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to be a very potent anti-oxidant [110]. A recent study shows that cysteine protease from Sesbania
grandiflora is very effective in exhibiting haemostatic and fibrinogenolytic activity [111].
Cardiometabolic diseases are highly treated with matrix metalloproteinases. Their mode of action is
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through modifying extracellular matrix components like collagen, gelatin and fibronectin [112]. The
overall therapeutic application of proteases is illustrated in figure 7.
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5.5 Protease Formulations

Researchers are focusing on preparing the protease enzymes into human friendly formulations for its
utmost usage in various application sectors. A few examples are discussed here. As proteases are very
dynamic in action, the enzyme is recommended as a skin care formulation for utility. Desquamation is
an important process that helps in maintaining the health of a skin. Physiological desquamation along
with functions of epidermal barrier, are completely dependent on water content of the body and also on
the presence of protease biomolecules and their inhibitors [113].
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Historically proteases have been used in the treatment of skin ulcers as reported by Ramundo and Gray
[114]. Recent advancements suggest that proteases could be used as a skin cleanser and also as a leave
on application in order to degrade the skin dirt which could be removed off from the skin by the
detergent component of the formulation [115]. Apart from skin care, proteases are being made into
formulations in order to improve the digestive ability of humans. An invention in United States patent
publications reports that, the usage of fungal protease along with effective excipients could act as a
health supplement for improving digestion and also equally active in raising pH level of blood which
has its own health benefits [116]. Beside skin care and digestive formulation applications, there are

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emerging protease based tooth-paste [117], protease capsules for wellness [118] and protease based
detergents [119]. Currently, with advanced techniques emerging in, researchers are emphasizing on

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synthesizing more formulations with protease as an active ingredient in all industries.

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6. A view on the statistics
Protease has been a great attraction to the researchers because of its widespread application potential.
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Over the period, huge number of research publications had been published in reputed journals to
establish the efficiency of proteases. The figure 8 and figure 9 shows the statistical data of proteases
retrieved from Scopus database. It includes year-wise publication on proteases for the past ten years;
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protease sources and their applications in percentages. The data showed that for the period of ten years
the research interest on proteases had kept increasing without a drop. It motivates the future researchers
and scientists to do study on proteases and produce more commercially viable products for the society.
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On analysing the patent scenario, there are around 215 patent publication over ten years, existing in
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India among which 21 were granted. The mentioned data are obtained from Indian Patent Advance
Search System – In PASS, Indian Patent Database. Globally, 5700 patent publications are available
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from 2008 to 2018 (source: worldwide database search – EPO). As per the World Bank Data, the Indian
Annual GDP (Gross Domestic Product) has been improved from 6.687 % in 2017 to 7.327 % in 2018
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(https://data.worldbank.org/country/india). The World Bank Forecasts Annual GDP for India in 2019
is 7.536%. It is a well-known fact that the food and beverages industry contributes a lot more to GDP
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growth. In order to achieve the increase in food sector product development, researchers should focus
on upbringing high yielding varieties through gene manipulation and also in synthesis of low cost and
high value products through economical downstream processing systems, which in turn could be
accessible and affordable to all the people in the society. On analysing the global scenario, America is
the leading consumer of enzymes and while considering Asian continent China is the larger supplier of
enzymes for various purposes followed by Japan. Certain surveys states that Western Europe would be
largest enzyme producer within a period of five to ten years [113] .

As we are in the concluding segment, it is more vital to think about future prospects of the protease
enzyme in medicines production sector. On analysing various reports, research publications and articles
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it was well understood that the downstream portion of the protease production involves enormous
money to be spent on it. The future research should be morally focused on developing economic
purification strategy of industrial proteases. The researchers would bring up on efficient substitutes to
column packing materials thereby cutting down the investment on downstream processing. To meet up
the affordable medical need of the people, scientists must explore on emergent pharmaceutical
applications of the protease enzyme. The finding of novel protease with economical downstream
processing costs and targeted therapeutical application, it is greatly achievable to serve the human
community to come over the medical struggles.

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7. Conclusion

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Proteases are creating a benchmark in every field with their potency and it is one of the most rapidly

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expanding area of exploration. Large numbers of researchers are focusing on engineering protease
producing genes for enhancing the quality and quantity of the product. A broad spectrum of human
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medical conditions is treated by protease enzyme and the success rate is enormous. In food industry,
the applications of protease are massive and measures are being taken to enhance the safety and value
of the food. Leather product manufacturers are purely dependant on enzyme processing for de-hairing
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in order to minimise the pollution created by the synthetic chemicals. The uncontrolled proteolysis
might be harmful in some ways but, with advancements in science and technology the methods to use
protease has been growing widespread thereby benefiting humankind.
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Figure legends

Figure 1: Graphical abstract

Figure 2: Brief outline of protease isolation and purification strategies
Figure 3: Stain-removal by protease embedded detergent molecule
Figure 4: Influential ingredients in detergent making
Figure 5: The leather tanning process
Figure 6: Steps in cheese making

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Figure 7: Overall therapeutic application of proteases
Figure 8: Statistical illustration of historical development of research on protease

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Figure 9: Statistical illustration of historical development of industrial applications of protease

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A systematic reconsideration on proteases

Poorani Gurumallesh, Kamalini Alagu, Baskar Ramakrishnan*, Shanmugaprakash Muthusamy

Figure 1: Graphical abstract

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A systematic reconsideration on proteases

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Figure 2: Brief outline of protease isolation and purification strategies

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Figure 3: Stain-removal by protease embedded detergent molecule

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Figure 4: Influential ingredients in detergent making

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Figure 5: The leather tanning process

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Figure 6: Steps in cheese making

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Figure 7: Overall therapeutic application of proteases

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Figure 8: Statistical illustration of historical development of research on protease

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Publications on extraction of protease from various sources
Source: SCOPUS database
40000

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35000
6248 5923 5860 5676
30000 6641

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6238
6047
25000 5489 6182 6049 6546 6571
5479 5549 5994
5362 5051 3973
20000 4376
3939
NU
3465 5029
15000 14707 14535 14594 14510
14278 14374
12708 13238
1000011648 12266 8568
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5000
5521 6264 6504 6791 6905 7250 7461 5781
4040 4559 4950
0
2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018
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Plant Animal Microbial rDNA

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Figure 9: Statistical illustration of historical development of industrial applications of protease

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